Liposome Composition

ABSTRACT

The present invention provides a novel liposome composition containing eribulin or its pharmacologically permissible salt, and its method of manufacture.

TECHNICAL FIELD

The present invention relates to a novel liposome composition containing eribulin or its pharmacologically permissible salt. The present invention also relates to a method of manufacture of the liposome composition.

BACKGROUND ART

Liposomes are microscopic closed vesicles having an internal phase enclosed by one or more lipid bilayers, and are capable of holding water-soluble material in the internal phase, and lipophilic material in the lipid bilayer. When entrapping an active compound in liposome, and delivering it to target tissue, how to entrap the active compound in the liposome with high efficiency, and how to secure stable retention of the active compound by the liposome constitute important issues.

When entrapping lipophilic compounds in liposome, a high entrapment ratio can be achieved relatively easily, but except in cases of compounds which have very high membrane affinity such as amphotericin B (the principal agent in the liposomal drug AmBisome), retention stability in blood plasma is ordinarily low, and it is difficult to obtain sufficient improvement in pharmacokinetics. With respect to methods for entrapping water-soluble compounds in liposome, there are various methods such as the lipid film method (Vortex method), reverse phase evaporation method, surfactant removal method, freeze-thaw method, and remote loading methods (pH gradient method, ion gradient method). However, it is only the remote loading methods that provide close to a 100% entrapment ratio; an entrapment ratio on the order of only 5 to 30% is obtained from the other methods.

As remote loading methods, those using a pH gradient and ammonium sulfate ion gradient are known. The pH gradient method, which is a remote loading method using a pH gradient, is a technique for incorporating compounds into liposome by using the movement of molecular/ionic dissociation equilibrium due to the pH of the target compound.

As one example of a compound entrapped in liposome by the pH gradient method, one may cite, for example, doxorubicin (DOX, pKa: 8.2). After preparing a liposome solution with a buffer solution of pH 4, the external phase of the liposome is replaced with a pH 7 buffer solution. In the case where DOX is added to this liposome solution, as the molecular DOX in the pH 7 solution is lipophilic, it migrates to the liposome membrane rather than to the aqueous phase. In the case where the DOX that has migrated to the liposome membrane further contacts the pH 4 internal phase of the liposome, it becomes ionic, and is incorporated into the internal phase of the liposome. In this way, DOX is transported from the external phase to the internal phase of liposome by a movement of dissociation equilibrium (see Non-patent Literature 1, Non-patent Literature 2, and Patent Literature 1).

A variety of techniques have been reported for improving this type of remote loading method. In Non-patent Literature 3, a technique is disclosed for improving the entrapment ratio of active compounds by adding ethanol together with the active compound to the external phase of the liposome, when the pH gradient method is conducted in liposome of special composition called cholesterol-free liposome.

In Patent Literature 2, in addition to the pH gradient, a technique is disclosed for improving the entrapment ratio of active compounds by having copper ions exist in the internal phase of the liposome.

Instead of a pH gradient in the pH gradient method, the ammonium sulfate method, which is a remote loading method using an ammonium sulfate ion gradient, is a technique for incorporating active compounds into the internal phase of liposome by using an ion gradient such as bivalent ammonium sulfate (see Non-patent Literature 1 and Patent Literature 3).

In addition to an ion gradient based on ammonium sulfate, Patent Literature 4 discloses a technique for incorporating active compounds into liposome by adding boronic acid together with the active compound to the external phase of the liposome

Instead of an ion gradient based on ammonium sulfate, Patent Literature 5 discloses a technique wherein, compared to the case where ammonium sulfate is used, the release rate of the active compound is improved by incorporating the active compound into liposome using an ion gradient of glucuronic acid anion.

Thus, from the standpoint of entrapment ratio, remote loading methods are excellent entrapment methods. However, in the case where remote loading methods are used, except for special cases such as Doxil (a liposome preparation of DOX) where the active compound entrapped in the internal phase of the liposome is crystallized, there is the problem that the active compound tends to leak from the liposome in blood plasma, and that retention stability of the active compound is low.

As described above, with conventional technical methods, the current situation is that it is difficult to achieve coexistence of a high entrapment ratio of the active compound in liposome and retention stability of the active compound in liposome.

PRIOR ART LITERATURE Patent Literatures

-   Patent Literature 1: U.S. Pat. No. 5,192,549, Specification -   Patent Literature 2: PCT international Publication WO 2006/037230,     Pamphlet -   Patent Literature 3: U.S. Pat. No. 5,316,771, Specification -   Patent Literature 4: U.S. Pat. No. 6,051,251, Specification -   Patent Literature 5: PCT International Publication WO 2005/046643,     Pamphlet

Non-Patent Literatures

-   Non-patent Literature 1: Yasuyuki Sazuka, “Liposome Preparation     Method,” “New Developments in Liposome Application: Toward the     Development of Artificial Cells” (Kazunari Akiyoshi, Shigeru Tsujii,     editorial supervision)” NTS, (2005), pp. 33-37. -   Non-patent Literature 2: Mayer L D et al., Biochimica et Biophysica     Acta, (1986), 857: pp. 123-426. -   Non-patent Literature 3: N. Dos Santos et al Biochimica et     Biophysica Acta (2004), 1661(1): pp. 47-60.

OUTLINE OF THE INVENTION Problem to Be Solved by the Invention

The object of the present invention is to provide a liposome composition with a high entrapment ratio and retention stability of the active compound.

Means for Solving the Problem

As a result of diligent research aimed at solving the aforementioned problems, the present inventors discovered, with respect to a liposome composition whose active compound is eribulin or its pharmacologically permissible salt, that the entrapment ratio and retention stability of the active compound in the liposome composition are extremely high, thereby perfecting the present invention.

Namely, the present invention is as follows.

(1)

A liposome composition containing liposome, and containing an active compound in the liposome internal phase, wherein the active compound is eribulin or its pharmacologically permissible salt.

(2)

The liposome composition according to 1, wherein the liposome composition is in a solid or a liquid form.

(3)

The liposome composition according to 1 or 2, wherein the liposome internal phase further contains ammonium salt.

(4)

The liposome composition according to 3, wherein the concentration of the aforementioned ammonium salt is 10 mM or higher.

(5)

The liposome composition according to any one of 1 to 4, wherein the liposome internal phase further contains salt, acid, base and/or amino acid.

(6)

The liposome composition according to 5, wherein the concentration of the aforementioned salt is 1 to 300 mM.

(7)

The liposome composition according to 5 or 6, wherein the concentration of the aforementioned acid is 1 to 300 mM.

(8)

The liposome composition according to any one of 5 to 7, wherein the concentration of the aforementioned amino acid is 1 to 300 mM.

(9)

The liposome composition according to any one of 5 to 8, wherein the concentration of the aforementioned base is 1 to 300 mM.

(10)

The liposome composition according to any one of 1 to 9, wherein the concentration of the aforementioned active compound is 0.01 to 300 mg/mL.

(11)

The liposome composition according to any one of 1 to 10, wherein the aforementioned active compound is eribulin mesylate.

(12)

The liposome composition according to any one of 1 to 11, wherein the liposome internal phase further contains ammonium sulfate, citric acid, and an active compound.

(13)

The liposome composition according to any one of 1 to 12, wherein the liposome external phase contains sugar, electrolyte, and/or amino acid.

(14)

The liposome composition according to any one of 1 to 13, wherein the liposome external phase contains sugar or electrolyte, and amino acid.

(15)

The liposome composition according to 13 or 14, wherein the concentration of the aforementioned sugar is 2 to 20%.

(16)

The liposome composition according to any one of 13 to 15, wherein the concentration of the aforementioned amino acid is 1 to 300 mM.

(17)

The liposome composition according to any one of 1 to 16, wherein the liposome external phase contains sucrose or sodium chloride, and histidine.

(18)

The liposome composition according to any one of 1 to 17, wherein the aforementioned liposome internal phase does not substantially contain cyclodextrin.

(19)

The liposome composition according to any one of 1 to 18, wherein the liposome contains hydrogenated phosphatidylcholine.

(20)

The liposome composition according to any one of 1 to 19, wherein the liposome contains cholesterol.

(21)

The liposome composition according to any one of 1 to 20, wherein the liposome contains methoxypolyethylene glycol condensate.

(22)

The liposome composition according to 21, wherein the aforementioned methoxypolyethylene glycol condensate is distearoylphosphatidyl ethanolamino polyethylene glycol condensate.

(23)

The liposome composition according to any one of 1 to 22, wherein the liposome contains hydrogenated phosphatidylcholine, cholesterol, and distearoylphosphatidyl ethanolamino polyethylene glycol condensate.

(24)

The liposome composition according to 23, which contains 10 to 80% of the aforementioned hydrogenated phosphatidylcholine, 1 to 60% of the aforementioned cholesterol, and 0 to 50% of the aforementioned distearoylphosphatidyl ethanolamino polyethylene glycol condensate.

(25)

The liposome composition according to any one of 1 to 24, wherein the liposome contains hydrogenated soy phosphatidylcholine, cholesterol, and polyethylene glycol 2000-phosphatidylethanolamine.

(26)

A method of manufacture of the liposome composition according to any one of 1 to 25, including:

a step in which a liposome dispersion liquid containing liposome is provided;

a step in which the aforementioned liposome dispersion liquid is mixed with the aforementioned active compound; and

a step in which the aforementioned active compound is introduced into the liposome internal phase of the aforementioned liposome dispersion liquid.

(27)

The method according to 26, wherein the aforementioned liposome dispersion liquid does not substantially contain ammonium salt in the liposome external phase.

(28)

The method according to 26 or 27, wherein the pH of the liposome external phase of the aforementioned liposome dispersion liquid is 3 to 10.

(29)

The method according to any one of 26 to 28, wherein the pH of the liposome external phase of the aforementioned liposome dispersion liquid is 7 to 10.

(30)

The method according to 28 or 29, wherein the aforementioned pH is the pH of the liposome external phase of the aforementioned liposome dispersion liquid in the step in which the aforementioned liposome dispersion liquid and the aforementioned active compound are mixed.

(31)

The method according to any one of 26 to 30, wherein the step in which the aforementioned liposome dispersion liquid is provided includes: a step in which a liposome preparatory solution is provided that contains liposome and that contains ammonium salt in the liposome internal phase and liposome external phase; and a step in which the liposome external phase of the aforementioned liposome preparatory solution is substituted or diluted.

(32)

The method according to 31, wherein the step in which the aforementioned liposome external phase is substituted or diluted is a step in which the pH of the liposome external phase is made higher than the pH of the liposome internal phase.

(33)

The method according to 31 or 32, wherein the step in which the aforementioned liposome external phase is substituted or diluted is a step in which the difference between the pH of the liposome internal phase and the pH of the liposome external phase is 1 to 5.

(34)

The method according to any one of 26 to 33, wherein the pH of the aforementioned liposome internal phase is 3 to 9.

(35)

The method according to any one of 26 to 34, wherein the pH of the aforementioned liposome internal phase is 4 to 9.

(36)

The method according to any one of 26 to 35, wherein the pH of the aforementioned liposome internal phase is 5 to 8.

(37)

The method according to any one of 26 to 36, wherein the liposome external phase is a solution that contains electrolyte in the step in which the aforementioned active compound is introduced.

(38)

The method according to any one of 26 to 37, wherein the aforementioned liposome dispersion liquid does not substantially contain cyclodextrin in the liposome internal phase.

(39)

The method according to any one of 26 to 38, which further contains a step in which the pH of the liposome external phase is neutralized.

Effect of the Invention

According to the present invention, it is possible to offer a novel liposome composition. The liposome composition of the present invention entraps an active compound in the liposome internal phase with a high degree of efficiency, and has a high retention stability of the active compound.

BRIEF DESCRIPTION OF THE DRAWINGS

(FIG. 1) shows in vitro changes in the concentration of eribulin mesylate in a liposome composition in rat blood plasma (37° C.).

(FIG. 2) shows the in vivo antitumor activity of eribulin mesylate due to liposome in FaDu cancer-bearing nude mice.

(FIG. 3) shows the in vivo antitumor activity of eribulin mesylate due to liposome in ACHN cancer-bearing nude mice.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention is specifically described by modes for carrying out the invention, but the present invention is not limited to the following modes for carrying out the invention, and may be carried out with a variety of modifications.

The contents disclosed in the literature referenced in the present invention are incorporated into the present invention as reference.

DEFINITIONS

“Liposome” means microscopic closed vesicles having an internal phase enclosed by lipid bilayer. In the present invention, liposome includes small single-membrane liposome (SUV: small unilamellar vesicle), large single-membrane liposome (LUV: large unilamellar vesicle), still larger single-membrane liposome (GUV: giant unilamellar vesicle), multilayer liposome having multiple concentric membranes (MLV: multilamellar vesicle), liposome having multiple membranes that are not concentric, but irregular (MVV: multivesicular vesicle), etc.

“Liposome internal phase” means an aqueous region enclosed in the lipid bilayer of the liposome, and is used with the same meaning as “internal water phase” and “liposome internal water phase.” “Liposome external phase” means the region not enclosed by the lipid bilayer of the liposome (that is, the region apart from the internal phase and the lipid bilayer) in the case where the liposome is dispersed in liquid.

“Liposome composition” means a composition that contains liposome and that further contains eribulin mesylate in the liposome internal phase. In the present invention, liposome composition includes both solid and liquid forms.

“Liposome dispersion liquid” means a composition containing liposome, and is a composition preceding the introduction of the active compound into the liposome internal phase.

“Liposome preparatory solution” means a composition containing liposome, and is a composition preceding adjustment of the liposome external phase for purposes of entrapping eribulin mesylate in the liposome internal phase.

“Liposome reagent” means a liposome dispersion liquid, in the case where it is in a liquid form. In the case where it is in a solid form, it means a reagent from which liposome dispersion liquid can be obtained by dissolution or suspension in a prescribed solvent. The solvent is described below. As described below, a solid liposome reagent can be obtained, for example, by drying a liposome dispersion liquid.

In the present specification, “the mixing of solid and liquid” includes the dissolution and suspension of the solid in the liquid, and mixing, dissolution and suspension are used in a mutually interchangeable manner. Similarly, solvent and dispersion medium are also used in a mutually interchangeable manner.

Moreover, the liposome composition, liposome dispersion liquid, liposome preparatory solution, and liposome reagent of the present invention do not substantially contain cyclodextrin. “To not substantially contain cyclodextrin” means that there is no addition of cyclodextrin. It is sufficient if cyclodextrin is not contained in a quantity in which improvement of the solubility (nominal solubility) of the active compound due to cyclodextrin is significantly observable, and even in the case where it is added in a quantity in which improvement in the solubility of the active compound is not significantly observable, it is not to be excluded from implementation of the present invention.

Furthermore, as a preferred mode of the present invention, “the liposome dispersion liquid not substantially containing ammonium salt in the liposome external phase” means that ammonium salt is not added to the liposome external phase of the liposome dispersion liquid. Addition of ammonium salt in a quantity which is within a range that can achieve the objective of the present invention is not to be excluded from implementation of the present invention. In the case where ammonium salt is contained in the liposome external phase of a liposome preparatory solution, it is possible to prepare a liposome dispersion liquid that does not substantially contain ammonium salt by substituting or diluting the liposome external phase of the liposome preparatory solution using a solution that does not substantially contain ammonium salt.

(Active Compound)

The active compound of the present invention is eribulin or its pharmacologically permissible salt (hereinafter sometimes referred to as “eribulin, etc.”). There are no particular limitations on the pharmacologically permissible salt so long as eribulin and salt are formed, whether inorganic acid salt or organic acid salt. For example, one may cite hydrochloric acid salt, sulfuric acid salt, citrate, hydrobromic acid salt, hydroiodine acid salt, nitric acid salt, bisulfate, phosphoric acid salt, super phosphoric acid salt, isonicotinic acid salt, ascetic acid salt, lactic acid salt, salicic acid salt, tartaric acid salt, pantotenic acid salt, ascorbic acid salt, succinic acid salt, maleic acid salt, fumaric acid salt, gluconic acid salt, saccharinic acid salt, formic acid salt, benzoic acid salt, glutaminic acid salt, methanesulfonic acid salt, ethanesulfonic acid salt, benzenesulfonic acid salt, p-toluenesulfonic acid salt, pamoic acid salt (pamoate), and so on. Preferable among these are hydrochloric acid salt, sulfuric acid salt, acetic acid salt, phosphoric acid salt, citrate, and mesylic acid salt, and most preferable of all is mesylic acid salt. That is, the preferable active compound of the present invention is eribulin mesylate. Moreover, as pharmacologically permissible salt of eribulin, it is acceptable to use eribulin and salt of aluminum, calcium, lithium, magnesium, calcium [sic], sodium, zinc, and diethanolamine. Eribulin or its pharmacologically permissible salt is the compound or its salt recorded in the pamphlet of PCT International Publication WO 99/65894 or U.S. Pat. No. 6,214,865 (the contents recorded in these patents are incorporated herein by reference), and have pharmacological action including antitumor action and antimitotic action. Eribulin or its pharmacologically permissible salt exhibits antitumor action with respect to melanoma, fibrosarcoma, monocytic leukemia, colon cancer, ovarian cancer, breast cancer, bone cancer, prostate cancer, lung cancer, and ras-transformed, fibroblasts.

However, as active compounds that can be combined with eribulin, etc., one may choose from among compounds used in the fields of medicines (including diagnostic drugs), cosmetic products, food products, and so on. With respect to active compounds, it is acceptable to combine one or more compounds other than eribulin, etc.

As active compounds, one may cite low-molecular compounds, etc. Among these, compounds used as antitumor agents, antibacterial agents, anti-inflammatory agents, anti-myocardial infarction agents, and contrast agents are suitable.

With respect to the molecular weight of the active compound, a range of 100 to 2000 is preferable, a range of 200 to 1500 is more preferable, and a range of 300 to 1000 is even more preferable. Within these ranges, the liposome membrane permeability of the active compound is generally satisfactory, and the present invention may be suitably applied.

The active compounds include water-soluble compounds and lipophilic compounds, and so long as they are more or less soluble in water or aqueous solvents, the present invention may be applied.

There are no particular limitations on antitumor agents in the present invention, and one may cite, for example, camptothecin derivatives such as irinotecan hydrochloride, nogitecan hydrochloride, exatecan, RFS-2000, lurtotecan, BNP-1350, Bay-383441, PNU-166148, IDEC-132, BN-80915, DB-38, DB-81, DB-90, DB-91, CKD-620, T-0128, ST-1480, ST-1481, DRF-1042, DE-310; taxane derivatives such as docetaxel hydride, docetaxel, pacritaxel, IND-5109, BMS-184476, BMS-188797, T-3782, TAX-1011, SB-RA-31012, SBT-1514, and DJ-927; iphosphamide, nimstine hydrochloride, carvocon, cyclophosphamide, dacarbazine, thiotepa, busulfan, melfaran, ranimustine, estramustine phosphate sodium, 6-mercaptopurine riboside, enocitabine, gemcitabine hydrochloride, carmfar, cytarabine, cytarabine ocfosfate, tegafur, doxifluridine, hydroxycarbamide, fluorouracil, methotrexate, mercaptopurine, fludarabine phosphate, actinomycin D, aclarubicin hydrochloride, idarubicin hydrochloride, pirarubicin hydrochloride, epirubicin hydrochloride, daunorubicin hydrochloride, doxorubicin hydrochloride, epirubicin, pirarubicin, daunorubicin, doxorubicin, pirarubicin hydrochloride, bleomycin hydrochloride, zinostatin stimalamer, neocarzinostatin, mitomycin C, bleomycin sulfate, peplomycin sulfate, etoposide, vinorelbine tartrate, vincrestine sulfate, vindesine sulfate, vinblastine sulfate, amrubicin hydrochloride, gefinitib, exemestane, capecitabine, TNP-470, TAK-165, KW-2401, KW-2170, KW-2871, KT-5555, KT-8391, TZT-1027, S-3304, CS-682, YM-511, YM-598, TAT-59, TAS-101, TAS-102, TA-106, FK-228, FK-317, E7070, (8E,12E,14E)-7-[(4-cycloheptypiperazine-1-yl)carbonyl]oxy-3,6,16,21-tetrahydroxy-6,10,12,16,20-pentamethyl-18,19-epoxytricosa-8,12,14-trien-11-olide (E7107), KRN-700, KRN-5500, J-107088, HMN-214, SM-11355, ZD-0473, etc. With respect to the compounds recorded as salts among the aforementioned antitumor agents, any salt is acceptable, and free bodies are also acceptable. With respect to compounds recorded as free bodies, any salt is acceptable.

There are no particular limitations on antibacterial agents, and one may cite, for example, amfotericine B, cefotiam hexyl, cephalosporin, chloramphenicol, diclofenac, etc. With respect to compounds of the aforementioned antibacterial agents, any salt is acceptable.

There are no particular limitations on anti-inflammatory agents, and one may cite, for example, prostaglandins (PGE1, PGE2), dexamethasone, hydrocortisone, pyroxicam, indomethacin, prednisolone, etc. With respect to compounds of the aforementioned anti-inflammatory agents, any salt is acceptable.

There are no particular limitations on anti-myocardial infarction agents, and one may cite, for example, adenosine, atenolol, pilsicamide, etc., and as contrast agents, one may cite, for example, iopamidol, ioxaglic acid, iohexyl, iomeprol, etc. With respect to compounds of the aforementioned anti-myocardial infarction agents and contrast agents, any salt is acceptable.

(Lipids)

It is preferable that the membrane constituents of the liposome of the present invention include phospholipids and/or phospholipid derivatives. As phospholipids and phospholipid derivatives, one may cite, for example, phosphatidyl ethanolamine, phosphatidyl choline, phosphatidyl serine, phosphatidyl inositol, phosphatidyl glycerol, cardiolipin, sphingomyelin, ceramide phosphorylethanolamine, ceramide phosphoryl glycerol, ceramide phosphoryl glycerol phosphate, 1,2-dimyristoyl-1,2-deoxyphosphatidyl choline, plasmalogen, phosphatidic acid, etc. It is also acceptable to combine one or more of these phospholipids and phospholipid derivatives.

There are no particular limitations on fatty-acid residues in the phospholipids and phospholipid derivatives, and one may cite, for example, saturated or unsaturated fatty-acid residue with a carbon number of 12 to 20. Specifically, one may cite acyl groups derived from fatty-acid such as lauric acid, myristic acid, palmitic acid, stearic acid, oleic acid, and linoleic acid. One may also use phospholipids derived from natural substances such as egg-yolk lecithin and soy lecithin, partially hydrogenated egg-yolk lecithin, (completely) hydrogenated egg-yolk lecithin, partially hydrogenated soy lecithin, and (completely) hydrogenated soy lecithin whose unsaturated fatty-acid residues are partially or completely hydrogenated, etc.

There are no particular limitations on the mixing amount (mole fraction) of the phospholipids and/or phospholipid derivatives that are used when preparing the liposome, but 10 to 80% relative to the entire liposome membrane composition is preferable, and 30 to 60% is more preferable.

With respect to membrane constituents, apart from phospholipids and/or phospholipid derivatives, the liposome of the present invention may also include sterols such as cholesterol and cholestenol as membrane stabilizers, fatty acids having saturated or unsaturated acyl groups with a carbon number of 8 to 22, and antioxidants such as α-tocopherol.

There are no particular limitations on the mixing amount (mole fraction) of these sterols that are used when preparing the liposome, but 1 to 60% relative to the entire liposome membrane composition is preferable, 10 to 50% is more preferable, and 30 to 50% is even more preferable.

Moreover, there are no particular limitations on the mixing amount (mole fraction) of the fatty acids, but 0 to 30% relative to the entire liposome membrane composition is preferable, 0 to 20% is more preferable, and 0 to 10% is even more preferable. With respect to the mixing amount (mole fraction) of the antioxidants, it is sufficient if an amount is added that can obtain the antioxidant effect, but 0 to 15% of the entire liposome membrane composition is preferable, 0 to 10% is more preferable, and 0 to 5% is even more preferable.

The liposome of the present invention may also contain functional lipids and modified lipids as membrane constituents.

As functional lipids, one may cite lipid derivatives retained in blood, temperature-sensitive lipid derivatives, pH-sensitive lipid derivatives, etc. As modified lipids, one may cite PEG lipids, sugar lipids, antibody-modified lipids, peptide-modified lipids, etc.

As lipid derivatives retained in blood, one may cite, for example, glycophorin, ganglioside GM1, ganglioside GM3, glucuronic acid derivatives, glutaminic acid derivatives, polyglycerin phospholipid derivatives, polyethylene glycol derivatives (methoxypolyethylene glycol condensates, etc.) such as N-[carbonyl-methoxy polyethylene glycol-2000]-1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine, N-[carbonyl-methoxy polyethylene glycol-5000]-1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine, N-[carbonyl-methoxy polyethylene glycol-750]-1,2-distearoyl-sn-glycero-3-phosphoethanolamine, N-[carbonyl methoxy polyethylene glycol-2000]-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (MPEG 2000-distearoyl phosphatidyl ethanolamine), and N-[carbonyl-methoxy polyethylene glycol-5000]-1,2-distearoyl-sn-glycero-3-phosphoethanolamine, which are condensates of phosphoethanolamine and methoxy polyethylene glycol. By having the liposome contain lipid derivatives with blood retention properties, it is possible to improve the blood retention of the liposome, because the liposome becomes difficult to capture in the liver, etc. as a foreign impurity.

As temperature-sensitive lipid derivatives, one may cite, for example, dipalmitoyl phosphatidylcholine, etc. By having the liposome contain temperature-sensitive lipid derivatives, it is possible to cause destruction of liposome at specific temperatures, and cause changes in the surface properties of the liposome. Furthermore, by combining this with an increase in temperature at the target site of the tumor, etc., it is possible to destroy the liposome at the target site, and release the active compound at the target site.

As pH-sensitive lipid derivatives, one may cite, for example, dioleoyl phosphatidyl ethanolamine, etc. By having the liposome contain pH-sensitive lipid derivatives, it is possible to promote membrane fusion of liposome and endosome when the liposome is incorporated into cells due to the endocytosis, and improve transmission of the active compound to cytoplasm.

As sugar lipids, antibody-modified lipids, and peptide-modified lipids, one may cite lipids that are bonded with sugars, antibodies, or peptides that are compatible with the target cells or target tissue. By using modified lipids, the liposome can be actively transmitted to the target cells or target tissue.

There are no particular limitations on the mixing amount (mole fraction) of lipid derivatives with blood retention properties used when preparing the liposome, but 0 to 50% of the entirety of liposome membrane constituent lipids is preferable, 0 to 30% is more preferable, and 0 to 20% is even more preferable.

(Liposome)

As mentioned above, liposome is a microscopic closed vesicle having an internal phase enclosed by a lipid bilayer.

Ideally, with respect to the liposome, a) it is preferable that the liposome has a barrier function that prevents leakage of eribulin, etc to the liposome external phase after the eribulin, etc. is once entrapped in the internal phase of the liposome. In the case where it is used as a medicine, it is preferable that the liposome exhibits in vivo stability, and that the liposome has a barrier function that prevents leakage of eribulin, etc to the liposome external phase in blood when the liposome is administered in vivo.

The composition of membrane constituents for liposome having such membrane permeability at a level allowing practical application can be appropriately selected by those skilled in the art according to the active compound, target tissue and the like by referencing as necessary the embodiments described below (Hiroshi Kikuchi, et. al, “Liposome I—Preparation Method and Assay Method—,” Cell Technology (1983), 2(9): pp. 1136-1149, and reference literature cited in said literature).

When used as a medicine, it is preferable that the eribulin, etc. be released from the liposome after the liposome reaches the target tissue, cells, or intracellular organelles. With respect to liposome, the membrane constituents themselves are ordinarily biodegradable, and ultimately decompose in target tissue or the like. It is thought that the entrapped eribulin, etc. is released in this manner. Moreover, it is also acceptable if the liposome itself is incorporated into cells.

Not only can the liposome composition be targeted to target tissue such as solid cancer, but it can also be used to transmit active compounds to hematological cancer and so on. It can also be used as a slow release formulation, controlled release formulation, etc. in blood.

The particle size of liposome can be set according to the objective. For example, when it is intended to transmit liposome to cancerous tissue or inflamed tissue by the EPR (Enhanced Permeability and Retention) effect as an injection product or the like, it is preferable that liposome particle size be 30 to 400 nm, and it is more preferable that the particle size be 50 to 200 nm. In the case where the intention is to transmit liposome to macrophage, it is preferable that liposome particle size be 30 to 1000 nm, and it is more preferable that the particle size be 100 to 400 nm. In the case where liposome composition is to be used as an oral preparation or trans-dermal preparation, the particle size of liposome can be set at several microns. It should be noted that (1) in normal tissue, vascular walls serve as barriers (because the vascular walls are densely constituted by vascular endothelial cells), and microparticles such as supermolecules and liposome of specified size cannot be distributed within the tissue. However, in diseased tissue, vascular walls are loose (because interstices exists between vascular endothelial cells), increasing vascular permeability, and supermolecules and microparticles can be distributed to extravascular tissue (enhanced permeability). Moreover, (2) the lymphatic system is well developed in normal tissue, but it is known that the lymphatic system is not developed in diseased tissue, and that supermolecules or microparticles, once incorporated, are not recycled through the general system, and are retained in the diseased tissue (enhanced retention)—this is called the EPR effect (Matsumura, Maeda, Cancer Research, (1986), 46: pp. 6387-6392). Consequently, it is possible to control pharmacokinetics by adjusting liposome particle size.

In the present invention, liposome particle size means the weight-average particle size according to the dynamic light scattering method (quasi-elastic light scattering method). Here, particle size is shown that is measured by dynamic light scattering instruments (e.g., Zetasizer Nano ZS model manufactured by Malvern Instruments Ltd. and ELS-8000 manufactured by Otsuka Electronics Co., Ltd.). The instruments measure Brownian motion of the particles, and particle size is determined based on established dynamic light scattering methodological theory.

There are no particular limitations on the solvent of the liposome internal phase, and one may cite, for example, buffer solutions such as phosphate buffer solution, citrate buffer solution, and phosphate-buffered physiological saline solution, physiological saline water, culture mediums for cell culturing, etc. As solvent, in the case where buffer solution is used, it is preferable that the concentration of buffer agent be 5 to 300 mM, and 10 to 100 mM is more preferable. There are no particular limitations on the pH of the liposome internal phase, but 3 to 11 is preferable, and 4 to 9 is more preferable.

(Liposome Composition)

A liposome composition is offered according to the present invention. The liposome composition contains liposome, and further contains eribulin, etc. in the liposome internal phase. As mentioned above, the liposome composition includes both a solid form and a liquid form. In the case where the liposome is in a solid form, it can be made into a liquid form by dissolving or suspending it in a prescribed solvent as described below. In the case where the liposome composition is frozen solid, it can be made into a liquid form by melting by leaving it standing at room temperature.

The concentration of liposome and the concentration of the active compound in the liposome composition can be appropriately set according to the liposome composition objective, formulation, etc. In the case where the liposome composition is a liquid formulation, the concentration of liposome as the concentration of all lipids constituting the liposome may be set at 0.2 to 100 mM, and preferably at 1 to 30 mM. The concentration (dosage) of active compound in the case where the liposome composition is used as a medicine is described below. With respect to the quantity of cyclodextrin in the liposome composition, it is preferable that it be less than a 0.1 mol equivalent relative to the eribulin, etc., and it is more preferable that it be less than the limit of detection.

In the liposome composition of the present invention, the eribulin, etc. may be apportioned to the lipid bilayer.

There are no particular limitations on the solvent (dispersion medium) of the liposome composition in the case where the liposome composition is a liquid formulation, and one may cite, for example, buffer solutions such as phosphate buffer solution, citrate buffer solution, and phosphate-buffered physiological saline solution, physiological saline water, and culture mediums for cell culturing. There are no particular limitations on the pH of the liposome external phase of the liposome composition, but 3 to 11 is preferable, and 4 to 9 is more preferable.

One may also add the following to the liposome composition: monosaccharides such as glucose, glactose, mannose, fructose, inositol, ribose, and xylose; disaccharides such as lactose, sucrose, cellobiose, trehalose, and maltose; trisaccharides such as raffinose and melizitose; polysaccharides such as cyclodextrin; and sugar alcohols such as erythritol, xylitol, sortibol, mannitol and maltitol; polyvalent alcohols such as glycerin, diglycerin, polyglycerin, propylene glycol, polypropylene glycol, ethylene glycol, diethylene glycol, triethylene glycol, polyethylene glycol, ethylene glycol monoalkylether, diethylene glycol monoalkylether, 1,3-butylene glycol. One may also use combinations of sugar and alcohol.

For purposes of stable long-term storage of the liposome that is dispersed in the solvent (dispersion medium), from the standpoint of physical stability including coagulation and so on, it is preferable to eliminate the electrolyte in the solvent (dispersion medium) as much as possible. Moreover, from the standpoint of chemical stability of the lipids, it is preferable to set the pH of the solvent (dispersion medium) from acidic to the vicinity of neutral (pH 3.0 to 8.0), and to remove dissolved oxygen through nitrogen bubbling.

There are no particular limitations on the concentration of the sugar or polyvalent alcohol contained in the liposome composition, but in a state where the liposome is dispersed in a solvent, for example, it is preferable that the concentration of sugar be 2 to 20% (W/V), and 5 to 10% (W/V) is more preferable. With respect to the concentration of polyvalent alcohol, 1 to 5% (W/V) is preferable, and 2 to 2.5% (W/V) is more preferable. These solvents can also be used as the liposome external phase in the liposome dispersion liquid, and by substituting or diluting the liposome external phase of the liposome preparatory solution with these solvents, it is possible to change the solutions of the liposome external phase into these solutions.

It is preferable that solid formulations of the liposome composition include, for example, monosaccharides such as glucose, glactose, mannose, fructose, inositole, ribose, and xylose; disaccharides such as lactose, sucrose, cellobiose, trehalose, and maltose; trisaccharides such as raffinose and melizitose; polysaccharides such as cyclodextrine; and sugar alcohols such as erythritol, xylitol, sorbitol, mannitol, and maltitol. More preferable are blends of glucose, lactose, sucrose, trehalose, and sorbitol. Even more preferable are blends of lactose, sucrose, and trehalose. By this means, solid formulations can be stably stored over long periods. When frozen, it is preferable that solid formulations contain polyvalent alcohols (aqueous solutions) such as glycerin, diglycerin, polyglycerin, propylene glycol, polypropylene glycol, ethylene glycol, diethylene glycol, triethylene glycol, polyethylene glycol, ethylene glycol monoalkylether, diethylene glycol monoalkylether and 1,3-butylene glycol. With respect to polyvalent alcohols (aqueous solutions), glycerin, propylene glycol, and polyethylene glycol are preferable, and glycerin and propylene glycol are more preferable. By this means, it is possible to stably store the solid formulation over long periods. Sugars and polyvalent alcohols may be used in combination.

(Method of Manufacture of Liposome Composition)

According to the present invention, a manufacturing method is provided for the manufacture of a liposome composition containing eribulin or its pharmacologically permissible salt. The method for manufacturing the liposome composition includes: a step in which a liposome dispersion liquid containing liposome is provided; a step in which the aforementioned liposome dispersion liquid is mixed with the aforementioned active compound (eribulin or its pharmacologically permissible salt); and a step in which the aforementioned active compound is introduced into the liposome internal phase of the aforementioned liposome dispersion liquid.

It is preferable that the step in which a liposome dispersion liquid containing liposome is provided include a step in which a liposome preparatory solution is provided, and a step in which the liposome external phase of the aforementioned liposome preparatory solution is substituted or diluted.

The liposome preparatory solution can be provided, for example, by preparing liposome in a solution containing ammonium salt. By preparing the liposome preparatory solution in a solution containing ammonium salt, it is possible to make a liposome dispersion liquid that also contains ammonium salt in the liposome internal phase.

There are no particular limitations on the solution containing ammonium salt that is used when preparing the liposome preparatory solution, and any solution containing ammonium salt may be used.

As ammonium salt, one may cite, for example, ammonium chloride, ammonium borate, ammonium sulfate, ammonium formate, ammonium acetate, ammonium citrate, ammonium tartrate, ammonium succinate, and ammonium phosphate. Ammonium sulfate, ammonium acetate, ammonium citrate, ammonium tartrate, and ammonium phosphate are preferable among these; ammonium sulfate, ammonium citrate, and ammonium tartrate are more preferable; and ammonium sulfate is most preferable.

One may use these ammonium salts in combinations of two or more.

The concentration of ammonium salt in the solution containing ammonium salt can be appropriately set according to the quantity of eribulin, etc. to be entrapped, and higher is better; 10 mM or more is preferable; 20 mM or more is more preferable; and 50 mM or more is even more preferable. With respect to the pH of the solution containing ammonium salt, 3 to 9 is preferable, 4 to 9 is more preferable from the standpoint of balancing entrapment ratio and stability, and 5 to 8 is even more preferable.

A pH adjuster may be used in order to adjust the pH of the solution containing ammonium salt. There are no particular limitations on the concentration of the individual pH adjusters in the solution containing ammonium salt, but 1 to 300 mM is preferable, and 5 to 100 mM is more preferable.

As the pH adjuster, one may cite, for example, amino acids such as arginine, histidine, and glycine; acids such as ascorbic acid, benzoic acid, citric acid, glutaminic acid, phosphoric acid, acetic acid, propionic acid, tartaric acid, carbonic acid, lactic acid, boric acid, maleic acid, fumaric acid, malic acid, adipic acid, hydrochloric acid, and sulfuric acid; salts of the aforementioned acids such as sodium salt, potassium salt, and ammonium salt; and alkaline compounds (base) such as tris-hydroxymethylamino methane, ammonia water (ammonia), sodium hydride, and potassium hydride. As pH adjusters, sodium hydride, hydrochloric acid, ammonia water, acetic acid, lactic acid, tartaric acid, succinic acid, citric acid, and phosphoric acid are preferable; sodium hydride, ammonia water, hydrochloric acid, acetic acid, citric acid, and phosphoric acid are more preferable; and sodium hydride, ammonia water, hydrochloric acid, citric acid, and phosphoric acid are even more preferable. The pH adjusters may be used in combinations of two or more of the ammonium salts. In addition, buffer solutions may also be used as pH adjusters, such as phosphate buffer solution, citrate buffer solution, and phosphate-buffered physiological saline solution.

As the liposome preparatory solution, it is best to use a solution that is obtained by preparing liposome without substantial inclusion of cyclodextrin. As the liposome preparatory solution, the liposome internal phase may also contain salt, acid, base, and/or amino acid. In this case, it is preferable that the liposome internal phase contain the active compound, ammonium salt, and acid. As the ammonium salt, ammonium sulfate may be cited as the preferred example; as the acid, citric acid may be cited as the preferred example.

With respect to liposome preparation, one may cite the lipid film method (Vortex method), reverse phase evaporation method, ultrasonic method, pre-vesicle method, ethanol injection method, French press method, cholic acid removal method, Triton X-100 batch method, Ca²⁺ fusion method, ether injection method, annealing method, freeze-thaw method, etc.

The various conditions (quantities of membrane constituents, temperature, etc.) in liposome preparation may be suitably selected according to the liposome preparation method, target liposome composition, particle size, etc. (see Op. cit, Kikuchi (1983), etc.).

The liposome particle size may be optionally adjusted as necessary. Particle size may be adjusted, for example, by conducting extrusion (extrusion filtration) under high pressure using a membrane filter of regular pore diameter. Particle size adjustment may be conducted at any timing during manufacture of the liposome composition of the present invention. For example, it may be conducted before adjustment of the liposome external phase in the liposome preparatory solution, after adjustment of the liposome external phase in the liposome preparatory solution, or after introduction of the active compound into the liposome internal phase. It is preferable to conduct the particle size adjustment before introducing the active compound into the liposome internal phase, and it is more preferable to conduct it before adjusting the liposome external phase in the liposome preparatory solution.

The liposome dispersion liquid can be obtained by substituting or diluting the external phase of the obtained liposome preparatory solution. The substitution or dilution of the liposome external phase may be conducted once, or a combination of various types of substitution or dilution methods may be conducted multiple times.

As a method for substituting the liposome external phase of the liposome preparatory solution, one may cite dialysis, centrifugal separation, and gel filtration. By substituting the liposome external phase, the present invention can be implemented so that the liposome external phase does not substantially contain cyclodextrin or ammonium salt. Moreover, by substituting or diluting the liposome external phase, it is possible to efficiently entrap eribulin or its pharmacologically permissible salt in the liposome internal phase.

Dialysis may be conducted, for example, using a dialysis membrane. As a dialysis membrane, one may cite a membrane with molecular weight cut-off such as a cellulose tube or Spectra/Por.

With respect to centrifugal separation, centrifugal acceleration may be conducted preferably to 100,000 g or higher, and more preferably to 300,000 g or higher. By substituting the liposome external phase by centrifugation, one may also conduct liposome concentration in conjunction with substitution of the liposome external phase.

Gel filtration may be carried out, for example, by conducting fractionation based on molecular weight using a column such as Sephadex or Sepharose.

As the solvent (dispersion medium) used when substituting and/or diluting the liposome external phase, one may cite, for example, sucrose solution, saline solution, and culture medium for cell culturing. By using these solvents, it is possible to prepare a stable liposome composition.

There are no particular limitations on the pH of said solvent, but a range of 2 to 11 may be set; 3 to 10 is preferable, 6 to 10 is more preferable, and 7 to 10 is even more preferable. As described below, a pH gradient may be used to introduce the eribulin, etc. into the liposome internal phase. In this case, the pH of the solvent may be set so that the liposome external phase attains the target pH.

A pH adjuster may be used in order to adjust the pH of said solvent. There are no particular limitations on the concentration of use, but 1 to 300 mM is preferable, and 5 to 100 mM is more preferable.

As the pH adjuster, one may cite, for example, amino acids such as arginine, histidine, and glycine; acids such as ascorbic acid, benzoic acid, citric acid, glutaminic acid, phosphoric acid, acetic acid, propionic acid, tartaric acid, carbonic acid, lactic acid, boric acid, maleic acid, fumaric acid, malic acid, adipic acid, hydrochloric acid, and sulfuric acid; salts of the aforementioned acids such as sodium salt, potassium salt, and ammonium salt; and alkaline compounds such as tris-hydroxymethylamino methane, ammonia water, sodium hydride, and potassium hydride. Sodium hydride, hydrochloric acid, histidine, tartaric acid, succinic acid, citric acid, and phosphoric acid are preferable; sodium hydride, hydrochloric acid, histidine, tartaric acid, citric acid, and phosphoric acid are more preferable; and sodium hydride, hydrochloric acid, histidine, and phosphoric acid are even more preferable.

In order to improve the entrapment ratio of eribulin or its pharmacologically permissible salt in liposome, the entrapment ratio can be increased by adding a solution (salt solution) containing electrolyte to the liposome external phase to increase ion intensity. There are no particular limitations on the electrolyte (salt) contained in the liposome external phase, but sodium chloride and potassium chloride are preferable, and sodium chloride is more preferable. Physiological saline solution can also be used. Moreover, as the liposome external phase of the liposome dispersion liquid or the like, sugar, electrolyte, and/or amino acid may be included, and sugar or electrolyte, and amino acid may also be included. As sugar, sucrose may be cited as the preferred example; as electrolyte, physiological saline solution and sodium chloride may be cited as preferred examples; and as amino acid, histidine may be cited as the preferred example.

It is preferable that the obtained liposome dispersion liquid not substantially contain cyclodextrin or ammonium salt in the liposome external phase and liposome internal phase, but in the present invention, eribulin or its pharmacologically permissible salt may be introduced into the liposome internal phase even in the case where cyclodextrin or ammonium salt has for some reason been added to the liposome external phase of the liposome dispersion liquid, and even when the liposome external phase of the liposome dispersion liquid contains cyclodextrin or ammonium salt.

With respect to the lipid concentration of liposome in the liposome dispersion liquid, 1 to 100 mM is preferable, and 1-50 mM is more preferable. Within these ranges, it is possible to suitably form a greater number of liposome particles without impairing the physical properties of the liposome dispersion liquid.

The liposome composition can be obtained by mixing the obtained liposome dispersion liquid and the active compound of eribulin, etc., and by introducing the active compound into the liposome internal phase of the liposome dispersion liquid. It is preferable that the step of introduction include a step in which the membrane permeability of the liposome is enhanced in the mixed solution of liposome dispersion liquid and the active compound. By this means, entrapment of the eribulin, etc. in the liposome can be accomplished in a shorter period of time. However, even if no particular operations are conducted for the purpose of enhancing the membrane permeability of the liposome after mixing of the liposome dispersion liquid and the eribulin, etc., it is possible to entrap the eribulin, etc. in the liposome if the required time is taken.

In the step in which eribulin or its pharmacologically permissible salt is mixed, it is possible to use a substance dissolved in a solvent or a solid substance as the eribulin, etc. There are no particular limitations on the solvent, and one may use, for example, a substance identical to the liposome external phase of the liposome dispersion liquid.

At one's option as necessary, it is possible to use a pH gradient in introducing the eribulin, etc. into the liposome internal phase. In this case, with respect to the pH of the liposome internal phase of the liposome dispersion liquid, 3 to 9 is preferable, 4 to 9 is more preferable, and 5 to 8 is even more preferable.

Moreover, it is possible to set the pH of the liposome external phase higher than the pH of the liposome internal phase to create a pH gradient. A pH gradient of 1 to 5 is preferable, and 2 to 3 is more preferable.

Furthermore, it is possible to increase the entrapment ratio in the liposome by bringing the pH of the liposome external phase closer to the vicinity of the pKa of the eribulin, etc. 7.5 to 12.5 is preferable, 8.5 to 11.5 is more preferable, and 9 to 10.5 is even more preferable (the pKa of eribulin mesylate is 9.6).

As the liposome preparatory solution, it is optimal to use a solution that is obtained by preparing liposome without substantial inclusion of cyclodextrin.

As a method of enhancing the membrane permeability of liposome in the obtained mixed solution, one may cite the method of heating the mixed solution, the method of adding a membrane fluidizer to the mixed solution, etc.

In the case where the mixed solution is heated, the active compound can generally be more efficiently introduced into the liposome internal phase by heating to higher temperatures. Specifically, it is preferable to set the temperature of heating taking into consideration the thermal stability of the active compound and the employed liposome membrane constituents. In particular, it is preferable that the temperature of heating be set to the phase transition temperature of the lipid bilayer membrane of the liposome or higher.

The “phase transition temperature” of the lipid bilayer membrane of liposome means the temperature at which heat absorption starts (the temperature when endothermic reaction begins) in differential thermal analysis of elevated temperatures conditions. Differential thermal analysis is a technique enabling analysis of the thermal properties of specimens by measuring the temperature differences of a specimen or reference substance as a function of time or temperature while changing the temperature of the specimen or reference substance. In the case where differential thermal analysis is conducted with respect to liposome membrane constituents, the liposome membrane components fluidize as temperature increases, and endothermic reaction is observed. As is widely known in this technical field, the temperature range in which endothermic reaction is observed greatly varies according to the liposome membrane components. For example, in the case where liposome membrane components consist of a pure lipid, the temperature range in which endothermic reaction is observed is extremely narrow, and endothermic reaction is often observed within a range of ±1° C. relative to the endothermic peak temperature. On the other hand, in the case where liposome membrane components consist of multiple lipids, and particularly in the case where liposome membrane components consist of lipids derived from natural materials, the temperature range in which endothermic reaction is observed tends to widen, and endothermic reaction is observed, for example, within a range of ±5° C. relative to the endothermic peak temperature (that is, a broad peak, etc. is observed). According to the present invention, it is thought that liposome membrane fluidization is increased, and membrane permeability of the active compound is increased by raising the temperature higher than the phase transition temperature of the liposome lipid bilayer membrane.

For example, although dependent on the thermal stability and so on of the active compound and the employed liposome membrane constituents, it is preferable to have a temperature range from the phase transition temperature of the liposome lipid bilayer membrane to +20° C. of the phase transition temperature; a temperature range from the phase transition temperature to +10° C. of the phase transition temperature is more preferable; and a temperature range from +5° C. of the phase transition temperature to +10° C. of the phase transition temperature is even more preferable.

The heating temperature is ordinarily 20 to 100° C.; 40 to 80° C. is preferable; and 45 to 65° C. is more preferable.

Specifically, in the case of a liposome membrane whose principal ingredients are dipalmitoyl phosphatidylcholine (phase transition temperature as simple substance: 41° C.) and cholesterol, although it also depends on the composition thereof, a heating temperature of 40 to 60° C. is ordinarily preferable, and 45 to 50° C. is more preferable. Moreover, in the case of a liposome membrane whose principal ingredients are hydrogenated soy phosphatidylcholine (HSPC; phase transition temperature as simple substance: 50 to 60° C.) and cholesterol, although it also depends on the composition thereof, a heating temperature of 50 to 70° C. is ordinarily preferable, and 55 to 65° C. is more preferable. However, these heating temperatures in no way limit the present invention.

In the heating step, there are no particular limitations on the time at which the tempera tire is maintained at or above the phase transition temperature, and this may be properly set within a range, for example, of several seconds to 30 minutes. Taking into consideration the thermal stability of the active compound and lipids as well as efficient mass production, it is desirable to conduct the treatment within a short time. That is, it is preferable that the elevated temperature maintenance period be 1 to 30 minutes, and 2 minutes to 5 minutes is more preferable. However, these temperature maintenance times in no way limit the present invention.

Moreover, as stated above, it is also possible to enhance liposome membrane permeability by adding a membrane fluidizer to the obtained mixed solution (that is, adding it to the external phase side of the liposome). As a membrane fluidizer, one may cite organic solvents, surfactants, enzymes, etc. that are soluble in aqueous solvents. More specifically, as organic solvents, one may cite, for example, monovalent alcohols such as ethyl alcohol and benzyl alcohol; polyvalent alcohols such as glycerin and propylene glycol; aprotic polar solvents such as dimethyl sulfoxide (HMSO). As surfactants, one may cite, for example, anionic surfactants such as fatty acid sodium, monoalkyl sulfate, and monoalkyl phosphate; cationic surfactants such as alkyl trimethyl ammonium salt; ampholytic surfactants such as alkyl dimethylamine oxide; and non-ionic surfactants such as polyoxyethylene alkylether, alkyl monoglyceryl ether, and fatty acid sorbitan ester. As enzymes, one may cite, for example, cholinesterase and cholesterol oxidase. Those skilled in the art may set the quantity of membrane fluidizer according to the composition of liposome membrane constituents, the membrane fluidizer, etc., and taking into consideration the degree of efficiency of entrapment of the active compound due to addition of the membrane fluidizer, the stability of the liposome, etc.

The manufacturing method of the liposome composition of the present invention may include a step of adjusting the liposome external phase pH of the obtained liposome composition after the above-mentioned introduction step.

The external phase pH to be adjusted is not particularly limited, but may be preferably 4 to 10, more preferably 5 to 9, and even more preferably neutral 6 to 8 from the standpoint of chemical stability of the phospholipid composing the liposome.

In addition, a step of drying the obtained liposome composition may be further included. That is, when using a liposome composition as a liquid formulation, the liposome composition in a liquid form obtained in the above-mentioned introduction step may be used without modification as the final liposome composition, or the liposome external phase in the liquid liposome composition obtained in the above-mentioned introduction step may be adjusted (replaced, etc.) to make a final liposome composition. When doing so, the adjustment of the liposome external phase may be carried out similarly to the adjustment of the liposome external phase in a liposome preparatory liquid. In the case where the liposome composition is a liquid formulation, it may be used without further modification.

Furthermore, in the case where the liposome composition is to be made into a solid preparation, the liquid liposome composition obtained in the above-mentioned introduction step may be dried to make the final solid liposome composition. Freeze drying and spray drying may be cited as examples of methods for drying the liposome composition. In cases where the liposome composition is a solid preparation, it may be dissolved or suspended in a suitable solvent and used as a liquid formulation. The solvent to use may be appropriately set according to the purpose of use, etc. for the liposome composition, and in the case of using the liposome composition as an injection product, for example, the solvent is preferably sterile distilled water. In the case of using the liposome composition as a medicine, the physician or patient may inject the solvent into a vial into which the solid preparation is entrapped, for example, to make the preparation at the time of use. In the case where the liquid liposome composition is a frozen solid preparation, it may be used as a liquid formulation by storing in a frozen state, and returned to a liquid state by leaving to melt at room temperature or by rapidly melting with heat at the time of use.

(Pharmaceutical Compositions, etc.)

The liposome composition of the present invention may be used as a curative medicine in the medical field. Specifically, the liposome composition of the present invention may be used as an antitumor pharmaceutical composition.

In the case where the liposome composition of the present invention is used as a pharmaceutical composition, the liposome composition may be administered by injection (intravenous, intra-arterial, or local injection), orally, nasally, subcutaneously, pulmonarily, or through eye drops, and in particular local injection to a targeted group of cells or organ or other such injection is preferable in addition to intravenous injection, subcutaneous injection, intracutaneous injection, and intra-arterial injection. Tablet, powder, granulation, syrup, capsule, liquid, and the like may be given as examples of the formulation of the liposome composition in the case of oral administration. Injection product, drip injection, eye drop, ointment, suppository, suspension, cataplasm, lotion, aerosol, plaster, and the like may be given as examples of formulations of the liposome composition in the case of non-oral administration, and an injection product and drip infusion agent are particularly preferable.

The dosage of the pharmaceutical composition differs markedly depending on the type of target disease, the type of the active compound, as well as the age, sex, and weight of the patient, the severity of the symptoms, along with other factors, but ordinarily, the daily dosage of eribulin or its pharmacologically permissible salt for adults is not particularly restricted, although eribulin mesylate, which is a suitable salt, is ordinarily 0.1 to 10 mg. Also, the administration may be divided into more than one dose per day. A liposome composition containing, for example, 0.01-300 mg/mL eribulin or its [typographical error] [sic: pharmacologically] permissible salt to the liposome internal phase may be administered as the liposome composition of the present invention.

(Kit)

According to the present invention, a kit is provided for preparing the liposome composition. The kit may be used to prepare the liposome composition as a medicine, which may be used by a physician in clinical setting or a patient.

The kit includes a liposome reagent. The liposome reagent may be either a solid or a liquid form. If the liposome reagent is in a liquid form, the above-mentioned liposome dispersion liquid may be used as the liposome reagent. Also, if the liposome reagent is in a solid form, the liposome reagent can be dissolved or suspended in an appropriate solvent to obtain the liposome dispersion liquid, and the above-mentioned liposome dispersion liquid can be dried to obtain the liposome reagent. Drying may be carried out similarly to the above-mentioned drying of the liposome composition. When using the kit, if the liposome reagent is in a solid form, the liposome regent can be dissolved or suspended in an appropriate solvent to make the liposome dispersion liquid. When doing so, the solvent is similar to the liposome external phase in the above-mentioned liposome dispersion liquid.

The kit of the present invention further contains eribulin or its pharmacologically permissible salt (eribulin mesylate is a suitable salt). The eribulin or its pharmacologically permissible salt may be either in a solid or liquid form (a state of dissolved or suspended in a solvent). When using the kit, if the eribulin or the like is in a solid form, it is preferable that it be dissolved or suspended in an appropriate solvent to make a liquid form. The solvent can be appropriately set according to the physical properties and the like of the eribulin or the like, and may be made similar to the liposome external phase in the above-mentioned dispersion liquid, for example. The kit of the present invention may include an active compound other than eribulin or its pharmacologically permissible salt.

In the kit, the liposome reagent and the active compound may be packaged separately, or they may be in solid forms and mixed together.

In the case where the liposome reagent is in a solid form, excluding cases of dissolving or suspending to form a liposome dispersion liquid as above, the kit may be used by carrying out a step similar to that of mixing the liposome dispersion liquid and the active compound and of introducing the active compound in the liposome internal phase of the liposome dispersion liquid in the manufacturing method of the above-mentioned liposome composition.

It is thereby possible to manufacture a liposome composition in which an active compound is introduced into the internal phase of the liposome reagent.

In the case where the liposome reagent and the active compound are both in solid forms and are packaged together, the mixture of the liposome reagent and the active compound is appropriately dissolved or suspended in a solvent. When doing so, the solvent is similar to the liposome external phase in the above-mentioned liposome dispersion liquid. It is thereby possible to form a state in which the liposome dispersion liquid and the active compound are mixed, after which use is made possible by carrying out other steps in the introduction of the active compound in the liposome internal phase of the liposome dispersion liquid in the manufacturing method of the above-mentioned liposome composition.

EMBODIMENTS

The present invention is specifically described by giving embodiments and comparative examples, but is not limited to the embodiments below.

Embodiment 1 Preparation of an Aqueous Solution for the Liposome Internal Phase

396.4 mg of ammonium sulfate and 189.1 mg of citric acid monohydrate were dissolved in pure water, and this was diluted to 15 mL to prepare 200 mM ammonium sulfate/60 mM aqueous citric acid. After adjusting 2.5 mL of the 200 mM ammonium sulfate/60 mM aqueous citric acid with aqueous ammonia to a pH of 5.5, the aqueous solution for the liposome internal phase was diluted to 5 mL with pure water.

<Preparation of the Liposome Preparatory Liquid>

After dissolving 317.9 mg of hydrogenated soybean phosphatidylcholine (manufactured by Lipoid), 116.0 mg of cholesterol (manufactured by Sigma), and 130.4 mg of polyethylene glycol 2000-phosphatidylethanolamine (manufactured by Genzyme, MPEG 2000-distearoyl phosphatidylethanolamine) in 10 mL of chloroform, this was accurately dispensed into three vials, after which the chloroform of one vial was removed under reduced pressure in a rotary evaporator to create a lipid film. 5 mL of the aqueous solution for the liposome internal phase was heated to approximately 60° C. and added to the obtained lipid film, and this was agitated to prepare a liposome preparatory liquid. After treating the liposome preparatory liquid with ultrasonic waves for 20 minutes, it was granulated with an extruder (manufactured by Lipex Biomembranes) heated to approximately 65° C. to obtain the liposome preparatory liquid. The particle size of the liposomes in the obtained liposome preparatory liquid was measured using a dynamic light scattering method, and all were 90 to 100 nm.

<Preparation of the Liposome Dispersion Liquid>

Using Sephadex G-50 columns, the obtained liposome preparatory liquid was eluted with 0.9% sodium chloride/10 mM histidine aqueous solution (pH=7.6), substituting the liposome external phase with the 0.9% sodium chloride/10 mM histidine aqueous solution. After substituting the liposome external phase, this was centrifuged for 30 minutes at 400,000×g. After the centrifuging, this was redispersed, and 0.9% sodium chloride/10 mM histidine aqueous solution was used to prepare a volume of 5 mL, obtaining the liposome dispersion liquid.

<Preparation of the Active Compound Solution>

The eribulin mesylate was dissolved in 0.9% sodium chloride/10 mM histidine aqueous solution to obtain 1 mg/mL eribulin mesylate.

<Preparation of the Liposome Composition>

0.5 mL of the liposome dispersion liquid and 0.5 mL of the eribulin mesylate solution were mixed in a 10-mL glass vessel, and this was incubated for 3 minutes in 55° C. water to obtain a liposome composition with eribulin mesylate introduced in the liposomes.

<Measurement of the Entrapment Ratio>

The entrapment ratio was determined as described below.

The liposome composition entrapping an active compound was ultracentrifuged for 30 minutes at 400,000×g. The active compound concentration in the filtrate was measured with HPLC, quantitating the amount of active compound not entrapped in the liposomes. The entrapment ratio was calculated using the formula below.

$\begin{matrix} {{{{Entrapment}\mspace{14mu} {ratio}\mspace{14mu} (\%)} = \frac{\begin{matrix} \begin{matrix} {{{{Qty}.\mspace{11mu} {of}}\mspace{14mu} {active}\mspace{14mu} {compound}\mspace{14mu} {in}\mspace{14mu} {total}\mspace{14mu} {{qty}.\; ({mg})}} -} \\ {{{Qty}.\mspace{11mu} {of}}\mspace{14mu} {active}\mspace{14mu} {compound}\mspace{14mu} {in}\mspace{14mu} {filtrate}\mspace{14mu} {after}} \end{matrix} \\ {{ultracentrifugation}\mspace{14mu} ({mg})} \end{matrix}}{{{Qty}.{\; \;}{of}}\mspace{11mu} {active}\mspace{14mu} {compound}\mspace{14mu} {in}\mspace{14mu} {total}\mspace{14mu} {{qty}.\; ({mg})}}} \times 100} & \left( {{Formula}\mspace{14mu} 1} \right) \end{matrix}$

The entrapment ratio of eribulin mesylate was 90.9%.

Embodiment 2 Preparation of the Aqueous Solution for the Liposome Internal Phase

Similarly to Embodiment 1, 264.3 mg of ammonium sulfate and 126.1 mg of citric acid monohydrate were dissolved in pure water, and a graduated flask was used to dilute this to 10 mL to prepare 200 mM ammonium sulfate/60 mM aqueous citric acid. Of this, 1 mL was taken and adjusted to a pH of 5.5 with ammonia water, after which this was diluted with pure water to 2 mL to prepare the aqueous solution for the liposome internal phase.

<Preparation of the Liposome Preparatory Liquid>

80 mg each of a lipid mixture (hydrogenated soybean phosphatidylcholine:cholesterol:polyethylene glycol 2000-phosphatidylethanolamine=58.6:19.2:22.2 (by weight)) was weighed, 2 mL of the aqueous solution for the liposome internal phase was heated to approximately 80° C. and added thereto, and this was agitated to prepare the liposome preparatory liquid. This liposome preparatory liquid was granulated using an extruder (manufactured by Lipex Biomembranes) heated to approximately 80° C. to obtain the liposome preparatory liquid.

<Preparation of the Liposome Dispersion Liquid>

The obtained liposome preparatory liquid was diluted to 10 mL with the 0.9% sodium chloride/10 mM histidine aqueous solution (pH=7.6), and this was centrifuged for 30 minutes at 400,000×g. After centrifuging, all of the filtrate was disposed. The precipitate was redispersed with the 0.9% sodium chloride/10 mM histidine aqueous solution, and a graduated flask was used to prepare 1 mL of liquid, obtaining the liposome dispersion liquid.

<Preparation of the Drug Solution>

Eribulin mesylate (eribulin mesylate) was dissolved in the 0.9% sodium chloride/10 mM histidine aqueous solution and 5 mg/mL eribulin mesylate solution was obtained.

<Preparation of the Liposome Composition>

0.96 mL of the liposome dispersion liquid and 0.24 mL of the eribulin mesylate solution were mixed in a 10-mL glass vessel, and this was incubated for 3 minutes in 60° C. water to obtain a liposome composition with eribulin mesylate introduced in the liposomes.

<Stability in Rat Blood Plasma>

0.2 mL of the prepared eribulin mesylate entrapped liposome and 1.8 mL of rat blood plasma were mixed, and this was shaken at 37° C. using a liquid phase incubator. Immediately after the preparation, sampling was performed at 6 hours, 12 hours, 24 hours, 48 hours and 72 hours after the shaking was begun, and the residual quantity of eribulin mesylate in the liposomes was measured with HPLC.

The measurement results are shown in FIG. 1. As can be seen in FIG. 1, it was indicated that the eribulin mesylate was stably retained in the blood plasma even over the long time span of 120 hours, and gradual release was possible.

Embodiment 3 Preparation of the Aqueous Solution for the Liposome Internal Phase

264.3 mg of ammonium sulfate and 126.1 mg of citric acid monohydrate were dissolved in pure water to obtain approximately 15 mL. After adjusting the pH to 7.0 with aqueous sodium hydroxide, this was diluted with pure water to 20 mL to prepare the aqueous solution for the liposome internal phase (100 mM ammonium sulfate/30 mM citric acid).

<Preparation of the Liposome Preparatory Liquid>

378 mg of a lipid mixture (hydrogenated soybean phosphatidylcholine cholesterol:polyethylene glycol 2000-phosphatidylethanolamine=58.6:19.2:22.2 (by weight)) was weighed, 10 mL of the above-mentioned aqueous solution for the liposome internal phase was heated to approximately 80° C. and added thereto, and this was agitated to prepare the liposome preparatory liquid. This liposome preparatory liquid was granulated using an extruder (manufactured by Lipex Biomembranes) provided with a 50-nm polycarbonate membrane filter and heated to approximately 80° C. to obtain the liposome preparatory liquid with a particle size of approximately 80 nm.

<Preparation of the Liposome Dispersion Liquid>

Using Sephadex G-50 columns, the obtained liposome preparatory liquid was eluted with 0.9% sodium chloride/10 mM histidine aqueous solution (pH=7.6), substituting the liposome external phase with the 0.9% sodium chloride/10 mM histidine aqueous solution. After substituting the liposome external phase, this was centrifuged for 30 minutes at 400,000×g. After centrifuging, this was redispersed with 96 mg/mL sucrose/10 mM histidine aqueous solution (pH=7.6), and 10 mL of the solution was diluted to 10 mL to obtain the liposome dispersion liquid.

<Preparation of the Drug Solution>

Eribulin mesylate was dissolved with the 96 mg/mL sucrose/10 mM histidine aqueous solution (pH=7.6) and 5 mg/mL eribulin mesylate solution was obtained.

<Preparation of the Liposome Composition>

9.6 mL of the liposome dispersion liquid and 1.2 mL of the eribulin mesylate solution were mixed in a 10-mL glass vessel, and sodium hydroxide was used to adjust the pH to 9.5. This was incubated for 3 minutes in 60° C. water to obtain a liposome composition with eribulin mesylate introduced in the liposomes. After cooling, chloride was used to adjust the pH to 7.5. Similarly to Embodiment 1, the entrapment ratio was measured and found to be 99%.

Embodiment 4 Preparation of the Aqueous Solution for the Liposome Internal Phase

Similarly to Embodiment 1, 100 mM ammonium sulfate/30 mM citric acid (pH=5.5) was prepared.

<Preparation of the Liposome Preparatory Liquid>

Hydrogenated soybean phosphatidylcholine, cholesterol, and polyethylene glycol 2000-phosphatidylethanolamine were weighed according to the quantities shown in Table 1 below. After dissolving each in 3 mL of chloroform, the chloroform was removed under reduced pressure in a rotary evaporator to create a lipid film. 10 mL of the prepared aqueous solution for the liposome internal phase was heated to approximately 80° C. and added to the obtained lipid film, and this was agitated to prepare a liposome preparatory liquid. This was granulated using an extruder (manufactured by Lipex Biomembranes) heated to approximately 80° C. to obtain the granulated liposome preparatory liquid. The particle size of the liposomes in the obtained liposome preparatory liquid was measured using a dynamic light scattering method, and Rp. 1 was 77 nm, Rp. 2 95 nm, Rp. 3 79 nm, and Rp. 4 128 nm.

TABLE 1 Hydrogenated soybean Polyethylene glycol 2000- Rp. phosphatidylcholine Cholesterol phosphatidylethanolamine 1 234 mg 76 mg 15 mg 2 234 mg 76 mg 15 mg 3 222 mg 73 mg 87 mg 4 222 mg 73 mg 87 mg

<Preparation of the Liposome Composition>

Similarly to Embodiment 1, the liposome dispersion liquid was obtained. Also, eribulin mesylate was dissolved in 0.9% sodium chloride/10 mM histidine aqueous solution, and 5 mg/mL eribulin mesylate was obtained.

4.8 mL of each of the liposome dispersion liquids and 0.6 mL of eribulin mesylate solution were mixed in 10-mL glass vessels, which were incubated for 3 minutes in 60° C. water to obtain liposome compositions with eribulin mesylate introduced in the liposomes. 24.6 mL of the 0.9% sodium chloride/10 mM histidine aqueous solution was added to each of the liposome compositions, and a 0.22-μm polyvinylidene fluoride (PVDF) filter was used for filtering and sterilization, obtaining an administration sample (eribulin mesylate concentration: 0.1 mg/mL). Similarly to Embodiment 1, the entrapment ratio was measured and confirmed to be at least 90% in each of the prescriptions.

Female nude mice (NU/NU, Charles River Laboratories Japan, Inc.) were subcutaneously inoculated with human melanoma LOX cells, and 11 or 12 days later, the samples were administered into the caudal veins so as to be 10 mL/kg (1.0 mg/kg for the eribulin mesylate). A blood sample was taken and tumor tissue extraction was carried out with a cardiac puncture at fixed periods after administration (15 minutes, 30 minutes, 1, 2, 4, 8, 12, 24, 36, and 48 hours) (n=3). The blood was sampled in a test tube containing heparin, and within 30 minutes of the sampling, the blood was separated by centrifuging at 1,500×g for 10 minutes at 4° C. to obtain the blood plasma. All of the tumor tissue was extracted, washed with PBS, and wiped with water-absorbent paper, and then the tissue weight was immediately weighed and recorded. The tissue was placed in a test tube and cooled in ice water, and then stored at −80° C. until analysis was carried out.

The eribulin mesylate in the blood plasma and in the tumor tissue was measured using LC/MS/MS.

The PK parameters were calculated using non-compartment model analysis software (WinNonlin version 5.0.1). The results of the blood plasma PK parameters and tumor tissue PK parameters of the eribulin mesylate are shown respectively in Table 2 and Table 3.

TABLE 2 Rp. 1-4 and eribulin mesylate blood plasma PK parameters in LOX cancer-bearing mice AUC₀₋₁ AUC_(0-∞) CL V_(ss) t_(1/2) MRT Ratio Prescription (ng · hr/mL) (ng · hr/mL) (mL/hr/kg) (mL/kg) (hr) (hr) 1 Rp. 1 253049 258274 3.87 43.99 8.7 11.4 707.1 Rp. 2 176148 177893 5.62 56.40 6.8 10.0 487.0 Rp. 3 228151 233067 4.29 48.93 8.4 11.4 638.1 Rp. 4 221494 230541 4.34 55.88 9.4 12.9 631.2 Eribulin 363.02 365.247 2420 8032 3.7 3.3 1.0 mesylate Ratio 1 = AUC_(plasma liposome)/AUC_(plasma eribulin mesylate)

TABLE 3 Rp. 1-4 and eribulin mesylate blood plasma PK parameters in LOX cancer-bearing mice C_([max]) T_([max]) AUC₀₋₁ AUC_(0-∞) t_(1/2) MRT TPI Ratio Prescription (ng/g) (hr) (ng · hr/mL) (ng · hr/mL) (hr) (hr) (mL/g) 2 Rp. 1 692.1 4.0 24960.7 34581.8 22.8 38.8 0.13 5.5 Rp. 2 1002.9 8.0 16759.6 22301.1 22.2 34.5 0.13 3.5 Rp. 3 3965.7 12.0 41643.7 46297.3 16.1 23.3 6.20 7.4 Rp. 4 1132.8 12.0 28377.4 45005.6 23.7 44.3 0.20 7.2 Eribulin 323.425 0.25 4649.521 6294.283 17.8 27.7 17.23 1.0 mesylate Ratio 2 = AUC_(tumor liposome)/AUC_(tumor eribulin mesylate)

From Table 2 and Table 3, it can be seen that the AUC of the blood plasma and tumor tissue is increased in comparison to the free eribulin mesylate in all four liposome compositions 1 to 4 and therefore, the tumor migration quantity and retention of the eribulin mesylate are improved.

Embodiment 5 Preparation of the Aqueous Solution for the Liposome Internal Phase

Similarly to Embodiment 1, 100 mM ammonium sulfate/30 mM aqueous citric acid (pH=5.5) was prepared.

<Preparation of the Liposome Preparatory Liquid>

221.8 mg of hydrogenated soybean phosphatidylcholine, 72.5 mg of cholesterol, and 86.9 mg of polyethylene glycol 2000-phosphatidylethanolamine were weighted. After dissolving them in 3 mL of chloroform, the chloroform was removed under reduced pressure in a rotary evaporator, and a lipid film was created. 10 ML of the created aqueous solution for the liposome internal phase were heated to approximately 80° C. and added to the obtained lipid film, and this was agitated to prepare a liposome preparatory liquid. This was granulated using an extruder (manufactured by Lipex Biomembranes) heated to approximately 80° C., and a granulated liposome preparatory liquid was obtained. When the particle sizes of the liposomes in the obtained liposome preparatory liquid were measured using a dynamic light scattering method, they were approximately 90 nm.

<Preparation of the Liposome Dispersion Liquid>

Using Sephadex G-50 columns, the obtained liposome preparatory liquid was eluted with 0.9% sodium chloride/10 mM histidine aqueous solution (pH=7.6), substituting the liposome external phase with the 0.9% sodium chloride/10 mM histidine aqueous solution. After substituting the liposome external phase, this was centrifuged for 30 minutes at 400,000×g. After the centrifuging, this was redispersed, and the 0.9% sodium chloride/10 mM histidine aqueous solution was used to prepare 10 mL of liquid, creating a liposome dispersion liquid.

<Preparation of the Drug Solution>

Eribulin mesylate was dissolved in the 0.9% sodium chloride/10 mM histidine aqueous solution and 1 mg/mL eribulin mesylate solution was obtained. Also, as administration samples of free bodies, the eribulin mesylate solution was diluted with the 0.9% sodium chloride/10 mM histidine aqueous solution, and a 0.22-μm PVDF filter was used for filtering and sterilizing to obtain administration samples (eribulin mesylate concentrations: 0.3 mg/mL and 0.4 mg/mL).

<Preparation of the Liposome Composition>

1.8 mL of the liposome dispersion liquid and 1.2 mL of the eribulin mesylate solution were each mixed in a 10 mL glass vessel, which was incubated for 3 minutes in 60° C. water to obtain a liposome composition with eribulin mesylate introduced in the liposomes. The obtained liposome composition was diluted with the in 0.9% sodium chloride/10 mM histidine aqueous solution, and a 0.22-μm PVDF filter was used for filtering and sterilizing to obtain an administration sample (eribulin mesylate concentration: 0.2 mg/mL). Similarly to Embodiment 1, the entrapment ratio was measured and confirmed to be at least 90%.

FaDu (obtained from the American Type Culture Collection), which is a human pharyngeal squamous cell carcinoma line, was cultured and grown in a 10% bovine fetal serum-containing MEM culture. The cells were separated from the flask using 0.05% Trypsin-EDTA solution and collected. After washing with PBS, the cells were suspended in PBS so as to be 5×10⁷ cells/mL and kept on ice. 0.1 mL of cell suspension liquid were subcutaneously injected in the right ventral portion of 6-week old nude mice (Charles River Laboratories Japan, Inc.). Each mouse was observed daily, and notes were made appropriately in cases where abnormal conditions were found. Calipers were used to measure the tumor size over time, and the tumor size was calculated based on the calculation formula: major axis×(minor axis squared)÷2. At the point when the tumor size was 100 to 200 mm³, the mice were separated into groups such that the average values of the tumor sizes and the mouse body weights were uniform among the test groups (five mice per test group), and the drug was administered into the caudal vein (0.2 mL/20 g; 3 times in 7-day intervals).

The resulting changes in average tumor volume after sample administration are shown in FIG. 2.

As shown in FIG. 2, a tumor-reducing effect was not obtained even at 4 mg/kg, which is the maximum tolerated dose for free bodies, because FaDu is a cell line with a low sensitivity to eribulin mesylate. Meanwhile, in the case of the liposome composite, a clear tumor-reducing effect was found even with the administration of 2 mg/kg, which is below the maximum tolerance dose, indicating that an extremely high pharmacological effect can be obtained even for types of cancers against which there has been no success with eribulin mesylate.

Embodiment 6 Preparation of the Aqueous Solution for the Liposome Internal Phase

Similarly to Embodiment 1, 100 mM ammonium sulfate/30 mM aqueous citric acid (pH=5.5) was prepared.

<Preparation of the Drug Solution>

Similarly to Embodiment 5, administration samples (eribulin mesylate concentrations: 0.2 mg/mL, 0.3 mg/mL, and 0.4 mg/mL) of free bodies were obtained.

<Preparation of the Liposome Composition>

Except for the use of the liposome internal phase solution aqueous solution [sic: aqueous solution for the liposome internal phase] prepared as described above, the liposome composition (eribulin mesylate concentration: 0.3 mg/mL) was obtained similarly to Embodiment 5. Similarly to Embodiment 1, the entrapment ratio was measured and found to be at least 90%.

ACHN (obtained from the American Type Culture Collection), which is a human renal cancer cell line, was cultured and grown in a 10% bovine fetal serum-containing MEM culture. The cells were separated from the flask using 0.05% Trypsin-EDTA solution and collected. After washing with PBS, the cells were suspended in PBS so as to be 5×10⁷ cells/mL and then kept on ice. 0.1 mL of cell suspension liquid were subcutaneously injected in the right ventral portion of 6-week old nude mice (Charles River Laboratories Japan, Inc.). Each mouse was observed daily, and notes made appropriately in cases where abnormal conditions were found. Calipers were used to measure the tumor size over time, and the tumor size was calculated based on the calculation formula: major axis×(minor axis squared)≧2. At the point when the tumor size was 150 to 200 mm³, the mice were separated into groups such that the average values of the tumor sizes and the body weights of mice were uniform among the test groups (five mice per test group), and the drug was administered into the caudal vein (0.2 mL/20 g; 3 times in 7-day intervals).

The results of the change in average tumor volume after sample administration are shown in FIG. 3.

As shown in FIG. 3, because ACHN is a cell line that is resistant to eribulin mesylate, no significant difference was found between any of the 2 mg/kg administration, 3 mg/kg administration, and 4 mg/kg (maximum tolerance dose) free body administration groups and the non-treated group 45 days after the start of sample administration. Meanwhile, in the liposome composition 3 mg/kg administration group, a tumor-growth suppression effect was found, and a significant minor tumor volume value was indicated for the untreated group and the free body administration groups 45 days after start of sample administration. As thus indicated, it is possible to delay the growth of a tumor by preparing a liposomal formulation for a tumor for which a therapeutic effect has never been obtained before with eribulin mesylate.

Embodiment 7 Preparation of the Aqueous Solution for the Liposome Internal Phase

The 12 types of aqueous solutions for the internal phase shown below in Table 4 were created.

<Preparation of the Liposome Preparatory Liquid>

120 mg of a lipid mixture (hydrogenated soybean phosphatidylcholine:cholesterol:polyethylene glycol 2000-phosphatidylethanolamine=58.6:19.2:22.2 (by weight)) was weighed into test tubes, and 3 mL of each sample of the aqueous solution for the internal phase was heated to 80° C.

This liposome preparatory liquid was granulated using an extruder heated to approximately 80° C., and the liposome preparatory liquid was obtained.

<Preparation of the Liposome Dispersion Solution>

Using Sephadex G-50 columns, the obtained liposome preparatory liquid was eluted with 0.9% sodium chloride/10 mM histidine aqueous solution, substituting the liposome external phase with the 0.9% sodium chloride/10 mM histidine aqueous solution.

After substituting the liposome external phase, this was centrifuged for 1 hour at 400,000×g and the filtrate was completely removed. The precipitate was resuspended with 96 mg/mL sucrose/10 mM histidine aqueous solution (pH=7.5) so as to be approximately 2 mL.

The particle size of the obtained liposome dispersion liquid was measured using a dynamic light scattering method, and all were approximately 80 nm

<Preparation of the Drug Solution>

Eribulin mesylate was dissolved in the 96 mg/mL sucrose/10 mM histidine aqueous solution, and 5 mg/mL eribulin mesylate solution was obtained.

<Preparation of the Liposome Composition>

The liposome dispersion liquid and the eribulin mesylate solution were mixed in a 10-mL glass vessel such that the eribulin mesylate was 0.2 mg/mL and the total lipid concentration was 16 mmol/mL. This was heated for 5 minutes at 60° C. to obtain a liposome composition with eribulin mesylate introduced into the liposomes.

<Measurement of the Entrapment Ratio>

The entrapment ratio was measured similarly to Embodiment 1, and the results are shown in Table 4. As can be seen from Table 4, regardless of which ammonium salt was used in the internal phase, the entrapment ratio of eribulin mesylate clearly improved. In particular, the improvement in the entrapment ratio was marked when using ammonium sulfate, ammonium citrate, ammonium phosphate, and ammonium tartrate.

TABLE 4 Osmotic Entrapment No. Composition pH pressure ratio (%) 1 50 mM of ammonium sulfate 7.5 (adjusted 300 mOsm 69.4 2 50 mM of sodium sulfate with hydro- (adjusted 7.2 3 50 mM of ammonium acetate chloric acid with 36.8 4 50 mM of sodium acetate or sodium sucrose) 10.2 5 50 mM of ammonium phosphate hydroxide) 45.8 6 50 mM of sodium phosphate 14.6 7 50 mM of ammonium citrate 65.8 8 50 mM of sodium citrate 8.7 9 50 mM of ammonium succinate 14.7 10 50 mM of sodium succinate 10.0 11 50 mM of ammonium tartrate 74.7 12 50 mM of sodium tartrate 11.6

Embodiment 8 Preparation of the Aqueous Solution for the Liposome Internal Phase

Similarly to Embodiment 7, the aqueous solution for the liposome internal phase was prepared from 100 mM ammonium sulfate/30 mM aqueous citric acid (pH=7.5).

<Preparation of the Liposome Preparatory Liquid>

Similarly to Embodiment 7, the above-mentioned aqueous solution for the liposome internal phase was used to prepare a liposome preparatory liquid.

<Preparation of the Liposome Dispersion Liquid>

Using Sephadex G-50 columns, the obtained liposome preparatory liquid was eluted with 0.9% sodium chloride/10 mM histidine aqueous solution, substituting the liposome external phase with the 0.9% sodium chloride/10 mM histidine aqueous solution.

After substituting the liposome external phase, this was centrifuged for 1 hour at 400,000×g, completely removing the filtrate. The precipitate was resuspended with 96 mg/mL/10 mM histidine aqueous solution (pH=7.5), the liposome external phase was substituted with 96 mg/mL sucrose/10 mM histidine aqueous solution (pH=7.5), and a liposome dispersion liquid was obtained. The particle size of the obtained liposome dispersion liquid was measured using a dynamic light scattering method, and it was approximately 80 nm.

The liposome dispersion liquid was dispensed into seven vials, and ammonium sulfate (adjusted to a pH of 7.5 using aqueous sodium hydroxide) of a known quantity was added to the liposome external phase such that the vials had the concentrations of Table 5, and a liposome dispersion liquid was obtained in which the ammonium sulfate in the liposome external phase was of a known concentration.

<Preparation of the Drug Solution>

Eribulin mesylate was dissolved in the 96 mg/mL sucrose/10 mM histidine aqueous solution and 5 mg/mL eribulin mesylate solution was obtained.

<Preparation of the Liposome Composition>

The liposome dispersion liquid and the eribulin mesylate solution were mixed in a 10-mL glass vessel such that the eribulin mesylate was 0.2 mg/mL and the total lipid concentration was 16 mM. This was heated for 5 minutes at 60° C. to obtain a liposome composition with eribulin mesylate introduced into the liposomes.

<Measurement of the Entrapment Ratio>

The entrapment ratio was measured similarly to Embodiment 1, and the results are shown in Table 5. This shows that if even 0.4 mM of ammonium sulfate is present in the liposome external phase, the entrapment ratio drops markedly, and there is almost no entrapment if 10 mM of ammonium sulfate is present.

TABLE 5 External phase ammonium sulfate Entrapment No. Internal water phase concentration (mM) ratio (%) 1 100 mM ammonium sulfate 0 90.4 2 30 mM citric acid 0.016 90.8 3 pH = 7.5 0.08 91.3 4 0.4 75.9 5 2 36.8 6 10 16.1 7 50 8.6

Embodiment 9 Preparation of the Aqueous Solution for the Liposome Internal Phase

Similarly to Example 7, the aqueous solution for the liposome internal phase was prepared from 100 mM ammonium sulfate/30 mM aqueous citric acid (pH=7.5).

<Preparation of the Liposome Preparatory Liquid>

Similarly to Example 7, the above-mentioned aqueous solution for the liposome internal phase was used to prepare the liposome preparatory liquid.

<Preparation of the Liposome Dispersion Solution>

Using Sephadex G-50 columns, the obtained liposome preparatory liquid was eluted with 0.9% sodium chloride/10 mM histidine aqueous solution, substituting the liposome external phase with the 0.9% sodium chloride/10 mM histidine aqueous solution.

After substituting the liposome external phase, this was centrifuged for 1 hour at 400,000×g and the filtrate completely removed. The precipitate was resuspended with 96 mg/mL sucrose/10 mM histidine aqueous solution (pH=7.5), the liposome external phase was substituted with the 96 mg/mL sucrose/10 mM histidine aqueous solution (pH=7.5), and the liposome dispersion liquid was obtained. The particle size of the obtained liposome dispersion liquid was measured using a dynamic light scattering method, and it was approximately 80 nm.

<Preparation of the Drug Solution>

Eribulin mesylate was dissolved in the 96 mg/mL sucrose/10 mM histidine aqueous solution and 5 mg/mL eribulin mesylate solution was obtained.

<Preparation of the Liposome Composition>

The liposome dispersion liquid and the eribulin mesylate solution were mixed in a 10-mL glass vessel such that the eribulin mesylate was 0.2 mg/mL and the total lipid concentration was 16 mM. As shown in Table 6, each pH of the liposome external phase was adjusted using 1M sodium hydroxide aqueous solution. This was heated thr 5 minutes at 60° C. to obtain a liposome composition with eribulin mesylate introduced in the liposomes. Next, hydrochloric acid was used to adjust the pH of the external phase to 7.5.

<Measurement of the Entrapment Ratio>

The entrapment ratio was measured similarly to Embodiment 1, and the results are shown in Table 6. Along with the rise in the pH of the liposome external phase, the entrapment ratio of the eribulin rose substantially, reaching an entrapment ratio of nearly 100%.

TABLE 6 External Entrapment No. Internal water phase phase pH ratio (%) 1 100 mM ammonium sulfate 7.5 72.9 2 30 mM citric acid 8.0 79.8 3 pH = 7.5 8.5 86.4 4 9.0 92.8 5 9.5 98.5 6 10.0 100.0 7 10.5 99.3

Embodiment 10 Preparation of the Aqueous Solution for the Liposome Internal Phase

Similarly to Example 7, the aqueous solution for the liposome internal phase was prepared from 100 mM ammonium sulfate/30 mM aqueous citric acid (pH=7.5).

<Preparation of the Liposome Preparatory Liquid>

Similarly to Example 7, the above-mentioned aqueous solution for the liposome internal phase was used to prepare the liposome preparatory liquid.

<Preparation of the Liposome Dispersion Solution>

Using Sephadex G-50 columns, the obtained liposome preparatory liquid was eluted with 0.9% sodium chloride/10 mM histidine aqueous solution, substituting the liposome external phase with the 0.9% sodium chloride/10 mM histidine aqueous solution.

The liposome dispersion liquid was dispensed into four vials, which were centrifuged for 1 hour at 400,000×g, and the filtrate was completely removed. The precipitate of two of the vials was resuspended with 96 mg/mL sucrose/10 mM histidine aqueous solution (pH=7.5), and the liposome external phase was substituted with the 96 mg/mL sucrose/10 mM histidine aqueous solution (pH 7.5). The precipitate of the remaining two vials was resuspended with the 0.9% sodium chloride/10 mM histidine aqueous solution (pH=7.5), and the liposome external phase was substituted with the 0.9% sodium chloride/10 mM histidine aqueous solution (pH=7.5). The particle size of the obtained liposome dispersion liquids was measured using a dynamic light scattering method, and all were approximately 80 nm.

<Preparation of the Drug Solution>

Eribulin mesylate was dissolved in the 96 mg/mL sucrose/10 mM histidine aqueous solution and 5 mg/mL eribulin mesylate solution was obtained. Similarly, eribulin mesylate was dissolved in the 0.9% sodium chloride/10 mM histidine aqueous solution and 5 mg/mL eribulin mesylate solution was obtained.

<Preparation of the Liposome Composition>

The liposome dispersion liquid and the eribulin mesylate solution were mixed in a 10-mL glass vessel such that the eribulin mesylate was 0.2 mg/mL, and the total lipid concentration was 16 mM. The pH of the liposome external phase of one the two vials of the 96 mg/mL sucrose/10 mM histidine aqueous solution (pH=7.5) was adjusted to 9.5 by adding sodium hydroxide. Similarly, the pH of the liposome external phase of one the two vials of the 0.9% sodium chloride/10 mM histidine aqueous solution (pH=7.5) was adjusted to 9.5 by adding sodium hydroxide. These were heated for 5 minutes at 60° C. to obtain a liposome composition with eribulin mesylate introduced into the liposomes.

<Measurement of the Entrapment Ratio>

The entrapment ratio was measured similarly to Embodiment 1, and the results are shown in Table 7. Compared to the case where the liposome external phase is a sucrose, which is nonelectrolyte, the case of sodium chloride, which is an electrolyte, clearly obtains an extremely high entrapment ratio. In addition to the electrolyte effect, application of the pH gradient to make the liposome external phase an alkali achieved a 100% entrapment ratio.

TABLE 7 External phase Entrapment No. Internal water phase composition ratio (%) 1 100 mM ammonium sulfate 96 mg/mL sucrose 72.9 30 mM citric acid 10 mM histidine pH = 7.5 pH = 7.5 2 0.9% sodium chloride 95.9 10 mM histidine pH = 7.5 3 96 mg/mL sucrose 98.5 10 mM histidine pH = 9.5 4 0.9% sodium chloride 100.0 10 mM histidine pH = 9.5

The present application is based on a Japanese patent application (Japanese Patent Application 2009-082521) fled on Mar. 30, 2009, and a U.S. provisional patent application (61/164,653), and the contents thereof are incorporated herein as reference.

INDUSTRIAL APPLICABILITY

The present invention is capable of providing a method for manufacturing a liposome with a high retention stability of the active compound with a high entrapment ratio.

The liposome composition of the present invention is favorably used in therapeutic applications through the pharmacological effect of eribulin or its pharmacologically permissible salt. 

1-39. (canceled)
 40. A pharmaceutical composition comprising: an amount of eribulin and/or a pharmaceutically acceptable salt thereof, said amount being contained in the internal phase of a liposome; wherein the internal phase of said liposome comprises citric acid and/or a citrate salt and at least one ammonium (NH₄ ⁺) salt.
 41. The pharmaceutical composition according to claim 40, wherein said at least one said ammonium (NH₄ ⁺) salt is chosen from ammonium sulfate, ammonium citrate, and ammonium tartrate.
 42. The pharmaceutical composition according to claim 40, wherein the internal phase further comprises at least one pH adjuster.
 43. The pharmaceutical composition according to claim 42, wherein said at least one pH adjuster is chosen from a base.
 44. A method for preparing a pharmaceutical composition comprising eribulin and/or a pharmaceutically acceptable salt thereof comprising: (a) preparing a liposome, wherein the internal phase of said liposome comprises citric acid and/or a citrate salt and at least one ammonium (NH₄ ⁺) salt; (b) preparing a solution comprising eribulin and/or a pharmaceutically acceptable salt thereof; and (c) combining the liposome prepared in (a) with the solution prepared in (b) to obtain a liposome composition comprising eribulin and/or a pharmaceutically acceptable salt thereof entrapped in the internal phase of said liposome composition; wherein the entrapment achieved in (c) in said internal phase of said liposome composition results in an entrapment ratio of at least 60 to 100% of said eribulin and/or said pharmaceutically acceptable salt thereof.
 45. The method for preparing a pharmaceutical composition according to claim 44, further comprising: (d) adjusting the pH of the external phase of said liposome to a value sufficient to achieve entrapment of said eribulin and/or said pharmaceutically acceptable salt thereof in the internal phase of the liposome composition formed in (c); wherein said entrapment achieved in (c) in said internal phase of said liposome composition results in an entrapment ratio of at least 90 to 100% of said eribulin and/or said pharmaceutically acceptable salt thereof.
 46. The method for preparing a pharmaceutical composition according to claim 44, wherein in (a), said liposome further comprises at least one pH adjuster in the internal phase.
 47. The method for preparing a pharmaceutical composition according to claim 46, wherein said at least one pH adjuster is chosen from a base.
 48. A pharmaceutical liposome composition comprising eribulin and/or a pharmaceutically acceptable salt thereof obtained by a process comprising: (a) preparing a liposome, wherein the internal phase of said liposome comprises citric acid and/or a citrate salt and at least one ammonium (NH₄ ⁺) salt; (b) preparing a solution comprising eribulin and/or a pharmaceutically acceptable salt thereof; and (c) combining the liposome prepared in (a) with the solution prepared in (b) to obtain a liposome composition comprising eribulin and/or a pharmaceutically acceptable salt thereof entrapped in the internal phase of said liposome composition; wherein the entrapment achieved in (c) in said internal phase of said liposome composition results in an entrapment ratio of at least 60 to 100% of said eribulin and/or said pharmaceutically acceptable salt thereof.
 49. The pharmaceutical liposome composition according to claim 48, wherein said process further comprises: (d) adjusting the pH of the external phase of said liposome to a value sufficient to achieve entrapment of said eribulin and/or said pharmaceutically acceptable salt thereof in the internal phase of the liposome composition formed in (c); wherein said entrapment achieved in (c) in said internal phase of said liposome composition results in an entrapment ratio of at least 90 to 100% of said eribulin and/or said pharmaceutically acceptable salt thereof.
 50. The pharmaceutical liposome composition according to claim 48, wherein in (a), said liposome further comprises at least one pH adjuster in the internal phase.
 51. The pharmaceutical liposome composition according to claim 50, wherein said at least one pH adjuster is chosen from a base.
 52. A pharmaceutical composition comprising: (a) a liposome; (b) eribulin and/or a pharmaceutically acceptable salt thereof in the internal phase of said liposome; (c) a residue in the internal phase of said liposome, said residue originating from means internal to said liposome for achieving entrapment of said eribulin and/or the pharmaceutically acceptable salt thereof.
 53. The pharmaceutical composition according to claim 52, wherein said pharmaceutical composition further comprises a residue external to said liposome, said residue originating from means for adjusting the pH external to said liposome.
 54. The pharmaceutical composition according to claim 52, wherein said pharmaceutical composition further comprises a residue external to said liposome, said residue originating from means external to said liposome for achieving entrapment of said eribulin and/or said pharmaceutically acceptable salt thereof in the internal phase of the liposome composition.
 55. The pharmaceutical composition according to claim 53, wherein said pharmaceutical composition further comprises a residue in the internal phase of said liposome originating from means internal to said liposome for adjusting the pH in said internal phase of said liposome.
 56. The pharmaceutical composition according to claim 54, wherein said pharmaceutical composition further comprises a residue in the internal phase of said liposome originating from means internal to said liposome for adjusting the pH in said internal phase of said liposome.
 57. A pharmaceutical composition comprising: (a) a liposome comprising HSPC, cholesterol, and MPEG-DSPE; (b) eribulin and/or a pharmaceutically acceptable salt thereof in the internal phase of said liposome; (c) a residue in the internal phase of said liposome, said residue originating from means internal to said liposome for achieving entrapment of said eribulin and/or the pharmaceutically acceptable salt thereof; (d) said residue in the internal phase of said liposome further originating from means internal to said liposome for adjusting the pH in said internal phase of said liposome.
 58. The pharmaceutical composition according to claim 57, wherein said pharmaceutical composition further comprises a residue external to said liposome, said residue originating from means for adjusting the pH external to said liposome.
 59. The pharmaceutical composition according to claim 57, wherein said pharmaceutical composition further comprises a residue external to said liposome, said residue originating from means external to said liposome for achieving entrapment of said eribulin and/or said pharmaceutically acceptable salt thereof in the internal phase of the liposome composition.
 60. A pharmaceutical liposome composition comprising eribulin and/or a pharmaceutically acceptable salt thereof obtained by a process comprising: (a) preparing a liposome, wherein said liposome further comprises means internal to said liposome for achieving entrapment of eribulin and/or the pharmaceutically acceptable salt thereof in the internal phase of said liposome, and optionally wherein the pH of the internal phase of the liposome is adjusted with at least one pH adjuster; (b) preparing a solution comprising said eribulin and/or a pharmaceutically acceptable salt thereof; (c) combining the liposome prepared in (a) with the solution prepared in (b) to obtain a liposome composition comprising eribulin and/or a pharmaceutically acceptable salt thereof entrapped in the internal phase of said liposome composition; wherein the entrapment achieved in (c) in said internal phase of said liposome composition results in an entrapment ratio of at least 60 to 100% of said eribulin and/or said pharmaceutically acceptable salt thereof.
 61. The pharmaceutical liposome composition according to claim 60, wherein said entrapment ratio is at least 90 to 100%.
 62. The pharmaceutical liposome composition according to claim 60, wherein said process further comprises: (d) adding means for adjusting the pH external to said liposome.
 63. The pharmaceutical liposome composition according to claim 62, wherein said entrapment ratio is at least 90 to 100%.
 64. The pharmaceutical liposome composition according to claim 60, wherein said process further comprises: (d) providing means external to said liposome for achieving entrapment of said eribulin and/or said pharmaceutically acceptable salt thereof in the internal phase of the liposome composition.
 65. The pharmaceutical liposome composition according to claim 64, wherein said entrapment ratio is at least 90 to 100%.
 66. A method for preparing a pharmaceutical composition comprising eribulin and/or a pharmaceutically acceptable salt thereof comprising: (a) preparing a liposome, wherein said liposome further comprises means internal to said liposome for achieving entrapment of eribulin and/or said pharmaceutically acceptable salt thereof in the internal phase of said liposome, and optionally wherein the pH of the internal phase of the liposome is adjusted with at least one pH adjuster; (b) preparing a solution comprising said eribulin and/or said pharmaceutically acceptable salt thereof; (c) combining the liposome prepared in (a) with the solution prepared in (b) to obtain a liposome composition comprising eribulin and/or a pharmaceutically acceptable salt thereof entrapped in the internal phase of said liposome composition; and wherein the entrapment achieved in (c) in said internal phase of said liposome composition results in an entrapment ratio of at least 60 to 100% of said eribulin and/or said pharmaceutically acceptable salt thereof.
 67. The method for preparing a pharmaceutical composition according to claim 66, wherein said entrapment ratio is at least 90 to 100%.
 68. The method for preparing a pharmaceutical composition according to claim 66, further comprising: (d) adding means for adjusting the pH external to said liposome.
 69. The method for preparing a pharmaceutical composition according to claim 68, wherein said entrapment ratio is at least 90 to 100%.
 70. The method for preparing a pharmaceutical composition according to claim 65, further comprising: (d) providing means external to said liposome for achieving entrapment of said eribulin and/or said pharmaceutically acceptable salt thereof in the internal phase of the liposome composition.
 71. The method for preparing a pharmaceutical composition according to claim 70, wherein said entrapment ratio is at least 90 to 100%.
 72. A pharmaceutical composition comprising: (a) a liposome; (b) eribulin and/or a pharmaceutically acceptable salt thereof in the internal phase of said liposome; (c) a residue originating from means for obtaining a pH gradient between the internal and external phases of said liposome for achieving entrapment of said eribulin and/or the pharmaceutically acceptable salt thereof.
 73. The pharmaceutical composition according to claim 72, wherein the pH in the external phase of said liposome is more basic than the pH in the internal phase of said liposome.
 74. The pharmaceutical composition according to claim 72, wherein the pH in the external phase of said liposome is in a range of about 7.5 to about 12.5.
 75. A pharmaceutical composition comprising: (a) a liposome; (b) eribulin and/or a pharmaceutically acceptable salt thereof in the internal phase of said liposome; (c) means in the external phase of said liposome for establishing a pH gradient between the internal phase and external phase of said liposome, wherein the pH in the external phase of said liposome is more basic.
 76. A pharmaceutical composition comprising eribulin and/or a pharmaceutically acceptable salt thereof obtained by a process comprising: (a) preparing a liposome composition comprising eribulin and/or a pharmaceutically acceptable salt thereof entrapped in the internal phase of said liposome composition; and (b) adjusting the pH of the external phase of said liposome composition after preparing said liposome composition in (a) to be more basic than the internal phase of said liposome.
 77. The pharmaceutical composition according to claim 76, wherein the entrapment achieved in (a) in said internal phase of said liposome composition results in an entrapment ratio of at least 60 to 100% of said eribulin and/or said pharmaceutically acceptable salt thereof. 